Use of FVIIa or a tissue factor antagonist for regulating gene expression and cell migration or chemotaxis

ABSTRACT

The present invention relates to use of FVII and/or FVIIa and/or another TF agonist and/or FVIIai and/or another TF antagonist in therapeutic treatment of pathological conditions that can be related to cell migration or treated by specific regulation of cell migration or chemotaxis. The invention also relates to the use of of FVII and/or FVIIa and/or another TF agonist and/or FVIIai and/or another TF antagonist in therapeutic treatment of pathological conditions that can be related to regulation of expression of at least one gene in a cell, e.g., Cyr61 gene.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of PCT/DK00/00401 filed onJul. 14, 2000, and claims priority under 35 U.S.C. 119 of Danishapplication no. PA 1999 01117 filed on Aug. 12, 1999, Danish applicationno. PA 1999 01023 filed on Jul. 14, 1999, and U.S. provisionalapplication No. 60/148,300 filed on Aug. 11, 1999, the contents of whichare fully incorporated herein by reference.

FIELD OF INVENTION

[0002] A novel cell regulating activity of coagulation factor VII (FVII)or a tissue factor antagonist such as, for example, inactivatedcoagulation factor VIIa (FVIIai) of cells expressing tissue factor (TF)has been described. The present invention relates to a method forregulating cell migration or chemotaxis by contacting the cell withFVIIa or another TF agonist, or FVIIai or another TF antagonist anddetermining the migration of said cell. The invention also relates tothe use of FVIIa or another TF agonist, or FVIIai or another TFantagonist for the preparation of a medicament for regulation of cellmigration in a patient. Moreover the present invention relates to amethod of treatment, and a method of detecting the activity ofcompounds, in particular drug candidates that interact with cellmigration.

BACKGROUND OF THE INVENTION

[0003] The extrinsic pathway of blood coagulation is initiated whenFVIIa circulating in plasma binds to the integral-membrane protein,tissue factor (TF). The role of TF in blood coagulation has beenextensively studied. The involvement of FVIIa as a proteolytic enzyme inthe blood coagulation cascade is believed to be confined to theextracellular leaflet of TF expressing cells. An intracellular activityof FVIIa was first implied when the sequence of TF showed homology tothe cytokine/interferon- or heamatopoietic receptor superfamily. Thesubclass I of the heamotopoietic receptor family includes receptors forgrowth hormone, prolactin, interleukins 1 to 7, granulocyte-macrophagecolony stimulating factors, eiythropoitin and thrombopoitin. Subclass IIincludes TF and receptors for interferon a and b.

[0004] The resemblance of TF to this class of receptors was furthersubstantiated with the appearance of the crystal structure.Characteristic of this class of cytokine receptors that includesreceptors for interferon b and g and IL-10 is that their activation leadto rapid tyrosine phosphorylation of the receptors themselves, as wellas a subset of intracellular proteins. Within minutes after the initialtyrosine phosphorylation an array of mitogen-activated (Ser/Thr) kinases(MAPK) is activated. These kinases are arranged in several parallelsignalling pathways. Thorough studies of the putative intracellularsignalling capacity of FVIIa have shown that it induce mobilisation ofintracellular free calcium (Ca²⁺) in the human bladder carcinoma cellline, J82, which constitutively express TF and in umbelical veinendothelial cells which were pre-treated with interleukin-1 to expressTF, but have failed to show any cytokine-like activation ofintracellular tyrosine kinases. In conclusion FVIIa is believed, in a TFdependent manner, to induce mobilisation of intracellular Ca²⁺ throughactivation of phospholipase C. The mechanism by which FVIIa activatesphospholipase c is not known, but tyrosine kinase activation hasspecifically been ruled out.

[0005] Recent reports from a number of laboratories indicate that TF mayinfluence an array of important biological functions other thancoagulation., such as angiogenesis, embryo vascularization and tumormetastasis. At present, however, it is unclear how TF contributes tothese biological processes. The extracellular domain of TF consists oftwo fibronectin-type III-like modules, as in the typical class IIcytokine receptor extracellular domain, raising the possibility that TFmay play a role in signal transduction, the primary function of cytokinereceptor. However, TF has a very short cytoplasmic domain (only 21 aminoacid residues in length) and lacks membrane-proximal motifs that mediatebinding of the non-receptor Janus kinases (Jaks) that are essential forcytokine receptor signaling. Nonetheless, several biochemical findingssuggest a signal transduction function for TF. Analysis ofthe human TFprotein sequence revealed a putative phosphorylation site in thecytoplasmic domain, which is conserved in mouse, rat and rabbit TF.Specific serine residues in the cytoplasmic tail of TF arephosphorylated in cells following stimulation with protein kinase Cactivator. The human TF cytoplasmic tail is phosphorylated in vitro atmultiple sites when incubated with lysates of U87-MG cells. A potentialrole for the TF cytoplasmic domain in signal transduction is alsoindicated in studies that showed prometastatic function of TF iscritically dependent on the TF cytoplasmic domain. Further, TFcytoplasmic domain is shown to interact with actin-binding protein 280(ABP-280) and supports cell adhesion and migration through recruitmentof ABP-280 to TF-mediated adhesion contacts.

[0006] However, TF has also been shown to participate certain types ofcell signaling by serving as a cofactor for its physiological ligandFVIIa in an extracellular signaling by a proteolytic mechanism. Forexample, binding of FVIIa to cell surface TF is shown to induceintracellular Ca²⁺ oscillations in a number of TF expressing cells,transient phosphorylation of tyrosine in monocytes, activation of MAPkinase, alteration in gene expression in fibroblasts and enhancedexpression of urokinase receptor in tumor cells. Catalytically inactiveFVIIa (FVIIai) fails to induce many of the above signaling responses,from Ca²⁺ oscillations to MAP kinase activation and gene reduction, andit appears that the catalytic activity of FVIIa may be required for atleast some TF-FVIIa-mediated signal transduction. At present, not muchis known about signaling pathway(s) that are induced by proteolyticallyactive FVIIa and how the signals generated by FVIIa could contribute toangiogenesis and tumor metastasis.

[0007] To study temporal program of transcription that underlies theFVIIa-induced response, in the present study, we have examined theresponse of human fibroblasts to FVIIa using a cDNA microarray. The datarevealed that the cellular expression of several genes was detectablyaltered in fibroblasts upon exposure of to FVTIIa. One such gene isCyr61, a growth factor-inducible intermediate early gene, whose productis shown to promote cell adhesion, augment growth factor-induced DNAsynthesis and stimulate cell migration in fibroblasts and endothelialcells.

SUMMARY OF THE INVENTION

[0008] The present invention relates to usage of FVII and/or FVIIaand/or another TF agonist and/or FVIIai and/or another TF antagonist intherapeutic treatment of pathological conditions that can be related tocell migration or treated by specific regulation of cell migration orchemotaxis.

[0009] In another aspect the invention relates to the use of FVII and/orFVII and/or another TF agonist and/or FVIIai and/or another TFantagonist in therapeutic treatment of pathological conditions that canbe related to the regulation of expression of at least one gene in acell e.g. the Cyr61 gene.

[0010] In another aspect the invention relates to a method for inducingor enhancing cell migration, comprising the step of contacting said cellwith a tissue factor agonist

[0011] In one embodiment, the tissue factor agonist is FVII or FVIIa.

[0012] In another aspect the invention relates to a method of reducingor inhibiting cell migration, comprising the step of contacting the cellwith a tissue factor antagonist.

[0013] In one embodiment the tissue factor antagonist is modified FVII.

[0014] In one embodiment the cell is a human cell expressing tissuefactor, including fibroblasts, smooth muscle cells, tumour cells,haematopoietic cells and epithelial cells.

[0015] In one embodiment the modified factor VII is selected from factorVII modified with Dansyl-Phe-Pro-Arg chloromethyl ketone,Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethylketone, Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Pro-Argchloromethyl ketone, Dansyl-D-Glu-Gly-Arg chloromethyl ketone,Dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Argchloromethylketone.

[0016] In another aspect the invention relates to a method for inducingor enhancing wound healing in a patient, comprising administering tosaid patient an effective amount of a pharmaceutical compositioncomprising Factor VIIa or factor VII or another tissue factor agonist ora combination thereof.

[0017] In another aspect the invention relates to a method forinhibiting or reducing cell migration, invasion, migration-induced cellproliferation or angiogenesis in a patient having a disease or conditionassociated with undesired cell migration, invasion, migration-inducedcell proliferation or angiogenesis, comprising administering to saidpatient an effective amount of a pharmaceutical composition comprising atissue factor antagonist.

[0018] In one embodiment the disease or condition is primary tumourgrowth, tumour invasion or metastasis.

[0019] In another aspect the invention relates to the use of a tissuefactor agonist for the manufacture of a medicament for inducing orenhancing cell migration.

[0020] In another aspect the invention relates to the use of a tissuefactor antagonist for the manufacture of a medicament for reducing orinhibiting cell migration.

[0021] In another aspect the invention relates to a method of regulatingthe expression of at least one gene in a cell, comprising the step ofeither contacting said cell with a tissue factor agonist or contactingsaid cell with a tissue factor antagonist.

[0022] In one embodiment the gene is a gene belonging to the CCN genefamily.

[0023] In another embodiment the gene is selected from the groupconsisting of Cyr61, CTFG, dopamine D2 receptor, EST Incyte PD 395116 orP2U nucleotide receptor.

[0024] In one embodiment the gene is Cyr61 gene.

[0025] In one embodiment the regulation is inducing or enhancingexpression. In another embodiment the regulation is reducing orinhibiting expression.

[0026] In one embodiment FVII or FVIIa or another tissue factor agonistinduces or enhances gene expression and modified FVII or another tissuefactor antagonist reduces or inhibits gene expression, e.g. when thegene is a gene belonging to the CCN gene family, or the gene is selectedfrom the group consisting of Cyr61, CTFG, dopamine D2 receptor, ESTIncyte PD 395116 or P2U nucleotide receptor.

[0027] In another embodiment FVII or FVIIa or another tissue factoragonist reduces or inhibits gene expression, and modified FVII oranother tissue factor antagonist induces or enhances gene expression,e.g., when the gene is EST PD674714.

[0028] Diseased states, which may be treated, are pathologicalconditions such as, for example, atherosclerosis, tumour deposition,tumour growth, tumour invasion, metastasis, or angiogenesis. Otherstates that may be treated is, for example, healing of wounds includingregeneration of vessel walls and treatment of burns, or inflammation, orthe regulation of cell migration in vitro such as, for example, growingof tissue.

LIST OF FIGURES (fra 6011)

[0029] FIGS. 1A and 1B: Flow cytometric analysis of TF expression infibroblasts (1A). The cells were stained with either a murine monoclonalfluoresceinisothiocyanate (FITC)-conjugated mouse anti IgG-antibody(unfilled area) that was used as negative control or a monoclonalFITC-conjugated anti-tissue factor (TF) antibody (filled area). FIG. 1Bshows the procoagulant activity of fibroblasts. Fibroblasts with TFexpression generated a 10-fold increase in PCA compared to monocyteswithout TF expression.

[0030]FIG. 2A: Effects of FVIIa and FFR-FVIIa on PDGF-BB inducedchemotaxis in human fibroblasts. ν show the chemotactic response offibroblasts to different concentrations of PDGF-BB. Fibroblastsincubated with 100 nM FVIIa (λ) or 100 nM FFR-FVIIa (μ) migrated towardsdifferent concentrations of PDGF-BB. Results are means and SEM of threeseparate experiments. P-values less than 0.05,* was consideredstatistically significant (Student's t test).

[0031] FIGS. 3 A-D: The influence of different concentrations of FVIIaor FFR-FVIIa on PDGF-BB induced chemotaxis in fibroblasts. v showmigration of fibroblasts to different concentrations of PDGF-BB. Cellswere incubated with 12.5 (A), 25 (B), 50(C) and 100 (D) nM FVIIa (λ) orFFR-FVIIa (μ) and assayed in the Boyden chamber towards differentconcentrations of PDGF-BB. Results are mean and SEM of three differentexperiments. *=p<0.05, **=p<0.01 and ***=p<0.001 Student's t test.

[0032]FIG. 4A: A mixture of three monoclonal antibodies to TF blocks theeffects of FVIIa and FFR-FVIIa on PDGF-BB induced chemotaxis infibroblasts. ν show migration towards PDGF-BB of fibroblasts without TFantibodies, λ fibroblasts preincubated with TF antibodies and 100 nMFVIIa, and μ fibroblasts preincubated with TF antibodies and 100 nMFFR-FVIIa. Results are mean and SEM of three separate experiments.

[0033] FIGS. 5A and 5B: The influence of FXa on the chemotactic responseto PDGF-BB induced by FVIIa. Fibroblasts were preincubated with 200 nMTAP (FIG. 5A) (ν) or with 0.2-2 μM TAP (FIG. 5B) (ν) and then with 100nM FVIIa (λ). TAP was present during the entire experiments. Chemotaxiswas induced by different concentrations of PDGF-BB (5A) or by 0.1 ng/mlPDGF-BB (5B). Results are mean and SD of two separate experiments.

[0034]FIG. 6A: The influence of thrombin on the chemotactic response toPDGF-BB induced by FVIIa. Fibroblasts were preincubated with 5 U/mL(final concentration) Hirudin and then with 100 nM FVIIa. Hirudin waspresent during the entire experiments. Chemotaxis was induced bydifferent concentrations of PDGF-BB. ν show cells incubated with Hirudinalone and λ cells with Hirudin and FVIIa. Results are mean and SD of twoseparate experiments.

[0035]FIG. 7A: Effect of inhibition of PI3′-kinase on chemotaxis infibroblasts incubated with FVIIa. Cells were preincubated with varyingconcentrations of LY294002 for 30 min at 37° C., and then with 100 nMFVIIa (λ) or without FVIIa (ν). The inhibitor was present throughout thechemotaxis assay. Chemotaxis was induced by 0.1 ng/mL PDGF-BB. Resultsare mean and SD of two separate experiments.

[0036] FIGS. 8A AND 8B: Effect of inhibition of PLC on chemotaxis infibroblasts incubated with FVIIa. Cells were incubated with varyingconcentrations of U73122 (active PLC inhibitor) (8A) or U73343 (inactivecontrol) (8B) for 30 min at 37° C. before incubation with or without 100nM FVIIa, and then assayed in the Boyden chamber to a concentrationgradient of 0.1 ng/mL PDGF-BB. The agents were present during the entireexperiments. ν show cells with U73122 or U73343 alone, λ cells withU73122 or U73343 and FVIIa. Results are mean and SD of two separateexperiments.

[0037]FIG. 9: Release of inositol trisphosphate (IP₃) from fibroblastsstimulated with FVIIa, FFR-FVIIa alone or in combination with PDGF-BB.Cells were labelled over night with myo [³H] inositol, incubated with orwithout 100 nM FVIIa or FFR-FVIIa in the absence or presence of 10 ng/mLor 100 ng/mL PDGF-BB. Cells were then analysed for release in IP₃. Openbars show cells without FVIIa or FFR-FVIIa (control), hatched bars showcells with FFR-FVIIa, and black bars show cells incubated with FVIIa.

[0038]FIG. 10: Tyrosine phosphorylation of PLC-γ1 in response to PDGF-BBalone (control), FVIIa or FFR-FVIIa in combination with PDGF-BB. Cellswere incubated with 100 nM FVIIa or FFR-FVIIa for one hour, and thenwith or without PDGF-BB at indicated concentrations. Cells were lysedand tyrosine phosphorylation of PLC-γ1 detected as described in methods.

[0039]FIG. 1. Northern blot analysis confirming the data obtained withcDNA microarray assay. Ten μg of total RNA (from the same RNA samplesthat were used to isolate poly (A) RNA to generate probes forhybridization of cDNA microarray) were patiented to Northern blotanalysis and probed with ³²P-labeled Cyr61 (a partial length cDNA,obtained from Genomic Systems). Panel B. The hybridization signals arequantified with PhosphorImager (Molecular Dynamics).

[0040]FIGS. 2 and 2B. Time-dependentfactor VIIa-induced expression ofCyr61. Quiescent monolayers of WI-38 cells were treated with factor VIIa(5 82 g/ml) (2A) or PDGF-BB (10 ng/ml) (2B) for varying time periods.Total RNA (10 μg) was patiented to Northern blot analysis and probedwith radio labeled Cyr61. Ethidium bromide staining of 28S ribosomal RNAof the corresponding blot is shown in the bottom panel as RNA loadingcontrol.

[0041]FIG. 3. Dose-dependentfactor VIIa-induced expression of Cyr61.Quiescent monolayers of WI-38 cells were treated with varying doses offactor VIIa, 0, 0.1, 0.5, 2.0 and 5.0 μg/ml for 45 min. Total RNA (10μg) was patiented to Northern blot analysis and probed with radiolabeledCyr61. Ethidium bromide staining of 28S ribosomal RNA of thecorresponding blot is shown in the bottom panel as RNA loading control.

[0042]FIG. 4. Factor VIIa catalytic activity is required for the inducedexpression of Cyr61. Quiescent monolayers of WI-38 cells were treatedwith a control serum-free medium or serum-free medium cotaining factorVIIa (5 μg/ml) or active-site inactivated factor VIIa (VIIai, 5 μg/ml)for 45 min. Total RNA (10 μg) was patiented to Northern blot analysisand probed with radiolabeled Cyr61 . Ethidium bromide staining of 28Sribosomal RNA of the corresponding blot is shown in the bottom panel asRNA loading control.

[0043]FIG. 5. Factor VIIa-induced expression of Cyr61 is not abolishedby specific inhibitors offactor Xa and thrombin. Quiescent monolayers ofWI-38 cells were treated with control medium or the medium containingfactor VIIa (5 μg/ml; 100 nM for 45 min. Cells were preincubated with200 nM recombinant TAP lane 3) or hirudin (lane 4) for 30 min beforeexposure to factor VIIa for 45 min. Total RNA (10 μg) was patiented toNothern blot analysis and probed with radiolabeled Cyr61. Ethidiumbromide staining of 28S ribosomal RNA of the corresponding blot is shownin the bottom panel as RNA loading control.

[0044]FIG. 6. Effect of actinomycin-D and cycloheximide on factorVIIa-induced Cyr61 mRNA steady-state levels. Quiescent monolayers ofWI-38 cells were preincubated with a control vehicle, actinomycin D (10μg/ml) or cycloheximide (10 μg/ml) for 30 min before the cells wereexposed to factor VIIa (5 μg/ml) for 45 min. Total RNA (10 μg) waspatiented to Northern blot analysis and probed with radiolabeled Cyr61.Ethidium bromide staining of 28S ribosomal RNA of the corresponding blotis shown in the bottom panel as RNA loading control.

[0045]FIG. 7. Factor VIIa induces the expression of CTGF. Quiescentmonolayers of WI-38 cells were treated with factor VIIa (5 μg/ml) forvarying time periods. Total RNA (10 μg) was patiented to Northern blotanalysis and probed with radio labeled CTGF. Ethidium bromide stainingof 28S ribosomal RNA of the corresponding blot is shown As RNA loadingcontrol.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention relates to the use of FVII or FVIIa oranother TF agonist for the manufacture of a pharmaceutical compositionfor inducing or enhancing cell migration.

[0047] In a further aspect the present invention relates to the use ofFVII, FVIIa or another TF agonist for the manufacture of apharmaceutical composition for inducing or enhancing wound healing orangiogenesis.

[0048] In a still further aspect the present invention relates to theuse of FVIIai or another TF antagonist for the manufacture of apharmaceutical composition for inhibiting or preventing cell migration.

[0049] In one embodiment the cell migration is in a subject.

[0050] In a further aspect the present invention relates to the use ofFVIIai or another TF antagonist for the manufacture of a pharmaceuticalcomposition for inhibiting or preventing angiogenesis, metastasis,tumour growth or tumour invasion.

[0051] In a further aspect the present invention concerns a method forinducing or enhancing cell migration in a patient, which comprisesadministering an effective amount of FVII or FVIIa or another TF agonistto said patient.

[0052] In a still further aspect the present invention concerns a methodfor inhibiting or preventing cell migration in a patient, whichcomprises administering an effective amount of FVIIai or another TFantagonist to said patient.

[0053] In a particular embodiment the effective amount is a daily dosagefrom about 5 μg/kg/day to about 500 μg/kg/day.

[0054] In a further embodiment the TF antagonist comprises a modifiedFVIIa, for example, FFR-FVIIa.

[0055] The present invention provides a mechanism for an activity ofFVII and/or FVIIa that relates to stimulation of cell migration. Such amechanism provides the basis for establishing the involvement of FVIIand/or FVIIa in pathological conditions in which TF expressing cellslike endothelial cells, epithelial cells, fibroblasts, smooth musclecells and monocytes/ macrophages participate. The invention furthermoreprovides the basis for identifying specific pharmacological targets thatare useful for therapeutic intervention.

[0056] Thus, the present invention relates to usage of FVII and/or FVIIaand/or FVIIai in therapeutic treatment of pathological conditions thatcan be related to cell migration or treated by specific regulation ofcell migration.

[0057] In another aspect, the present invention relates to a method ofdetecting drug candidates that regulate cell migration, which methodcomprise

[0058] a) culturing a TF expressing cell;

[0059] b) measuring the migration of the cell;

[0060] c) incubating the cell with a drug candidate, and

[0061] d) measuring the migration of the incubated cell and determiningany change in the level of migration compared to the migration measuredin step b, such change being indicative of biologically active drugcandidate in said cell.

[0062] Generally, the blood components, which participate in what hasbeen referred to as the coagulation “cascade” are proenzymes orzymogens, enzymatically inactive proteins, which are converted toproteolytic enzymes by the action of an activator, itself an activatedclotting factor. Coagulation factors that have undergone such aconversion and generally referred to as “active factors”, and aredesignated by the addition of the letter “a” to the name of thecoagulation factor (e.g. factor VIIa).

[0063] The term “zinc-chelator” is intended to comprise a compound thatbinds to factor VIIa and induces replacement of calcium ions with zincions within factor VIIa, thereby inhibiting the activity of factor VIIaor tissue factor-factor VIIa complex (TF-FVIIa).

[0064] A suitable TF antagonist according to the invention may be azinc-chelating compound, e.g. a dihydroxamate or a dihydrazide with thehydroxamate or hydrazide groups located relative to each other in such aposition that they are able to chelate a zinc ion. The zinc-chelatingcompound acts in combination with FVIIa. Zn²⁺-ions exert theirinhibitory action in competition with a stimulatory effect of Ca²⁺-ions.It is predicted that Zn²⁺-ions displace Ca²⁺-ions from one or morecalcium binding site(s) within FVIIa. Zinc-chelating compounds, e.g.hydroxamates and hydrazides, are capable of acting as powerfullsupporters for binding of zinc ions in competition with calcium ions.Specific compounds thereby potentiate zinc inhibition of the activity ofthe factor VIIa/tissue factor complex. The activity of factor VIIa incomplex with tissue factor can be inhibited by a mechanism in which azinc chelator binds to factor VIIa and facilitates replacement of Ca²⁺with Zn²⁺. By this action the chelator exerts a modulatory effect on TFat the normal concentration of free Ca²⁺ and Zn²⁺ ions in the blood.

[0065] The term “FVII” or “factor VII” means “single chain” (zymogenic)coagulation factor VII. The term “Factor VIIa”, or “FVIIa” means “twochain” activated coagulation factor VII cleaved by specific cleavage atthe Arg152-Ile153 peptide bond. FVII and FVIIa may be purified fromblood or produced by recombinant means. It is evident that the practiceof the methods described herein is independent of how the purifiedfactor VIIa is derived and, therefore, the present invention iscontemplated to cover use of any factor VII or FVIIa preparationssuitable for use herein. Preferred are human FVIIa. FVII or FVIIa isalso intended to include FVII variants wherein one or more amino acidresidues has (have) been replaced.

[0066] The term “modified factor VII”, “inactivated FVII” or “FVIIai” isintended to mean FVIIa having at least one modification in its catalyticcentre, which modification substantially inhibits the ability ofmodified FVIIa to activate FX and FIX. The terms may be usedinterchangeably. Such modification includes amino acid substitution (orreplacement) of one or more of the catalytic triad residues Ser344,Asp142 and His193, and also includes modification of catalytic triadresidues with serine protease inhibitors such as organo-phosphorcompounds, sulfanylfluoride, peptide halomethyl ketone or azapeptide.FFR-FVIIa is one example of a FVIIai derivative obtained by blocking ofthe active centre of FVIIa with the irreversible inhibitor,D-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone (FFRcmk). Other suitable FVIIai derivates are inactivated FVIIa obtained orobtainable by blocking the active centre withL-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone,dansyl-L-phenylalanine-L-phenylalanine-L-argininine chloromethyl ketone,or dansyl-D-phenylalanine-L-phenylalanine-L-argininine chloromethylketone, Preferred is FFR-FVIIa (FVIIa inactivated by FFR cmk).

[0067] The term “protein kinase” is intended to indicate an enzyme thatis capable of phosphoiylating serine and/or threonine and/or tyrosine inpeptides and/or proteins.

[0068] The term “drug candidate” is intended to indicate any sample,which has a biological function or exerts a biological effect in acellular system. The sample may be a sample of a biological materialsuch as a microbial or plant extract, or it may be a sample containing acompound or mixture of compounds prepared by organic synthesis orgenetic techniques.

[0069] The term “TF agonist” comprises compounds inducing

[0070] a) signal transduction by direct binding to TF (e.g. FVIIa),

[0071] b) stimulation of MAPK cascade,

[0072] c) abrogation of MAPK inhibition (e.g. PTPase inhibitors), whichagonists are drug candidates as defined above.

[0073] The term “TF antagonist” comprises

[0074] a) reagents which compete with FVIIa for binding to TF withouttransmission, e.g. FVIIai,

[0075] b) reagents which bind to FVIIa and prevent binding to TF, e.g.Zn hydroxamate,

[0076] c) reagents which inhibit signal transduction by interfering withmembers of the MAPK cascade,

[0077] d) reagents which bind to FVIIa/TF and prevent transmission,

[0078] e) reagents which bind to FVIIa/TF/FX and prevent transmission,

[0079] f) reagents which block human factor X activation catalysed byhuman tissue factor/factor VIIa complex,

[0080] which antagonists are drug candidates as defined above.

[0081] The term “pharmacological targets” is intended to indicate aprotein that can alter the migration of TF expressing cells.

[0082] The term “reporter gene” is intended to indicate a DNA constructthat, when transcribed, produces a protein that can be detected.

[0083] The term “SRE promoter element” means a DNA sequence that bindstranscription factors induced by components present in serum.

[0084] The term “TF expressing cell” means any mammalian cell thatexpresses TF.

[0085] The term “protein phosphorylation” is intended to indicatephosphorylation of serine and/or threonine and/or tyrosine in peptidesand/or proteins.

[0086] Modulation or regulation of cell migration is defined as thecapacity of FVIIa or another TF agonist, or FVIIai or another TFantagonist to 1) either increase or decrease ongoing, normal orabnormal, cell migration, 2) initiate normal cell migration, and 3)initiate abnormal cell migration.

[0087] Modulation or regulation of gene expression encompasses thecapacity of FVIIa or another TF agonist, or FVIIai or another TFantagonist to 1) either increase or decrease ongoing, normal orabnormal, cell migration, 2) initiate normal cell migration, and 3)initiate abnormal cell migration.

[0088] Modulation or regulation of gene expression encompasses anincrease or decrease in any parameter of gene expression of at leastabout 1.5-fold, preferably at least about 2-fold, more preferably atleast about 3-fold, and most preferably at least about 5-fold. Usefulparameters of gene expression include, without limitation, rate oftranscription, level of mRNA accumulation, rate of synthesis of the geneproduct, and level of protein accumulation. Modulation of geneexpression may also be reflected in secondary indices known to those ofordinary skill in the art. Any measurable change in any of theseparameters indicates regulation of expression.

[0089] In this context, the term “treatment” is meant to include bothprevention of an adverse condition and regulation of an alreadyoccurring condition with the purpose of inhibiting or minimising thecondition. Prophylactic administration of FVIIa or another TF agonist,or FVIIai or another TF antagonist is thus included in the term“treatment”.

[0090] In this context, the term “one unit” is defined as the amount offactor VII present in 1 ml of normal plasma, corresponding to about 0.5μg protein. After activation 50 units correspond to about 1 μg protein.

[0091] In this context, the term “patient” is defined as any animal, inparticular mammals, such as humans. The term “subject” is usedinterchangeably with “patient” Abbreviations TF tissue factor FVIIfactor VII in its single-chain, unactivated form FVIIa factor VII in itsactivated form RFVIIa recombinant factor VII in its activated formFVIIai modified (inactivated) factor VII FFR-FVIIai factor VIIinactivated by reaction with D-Phe-L-Phe-L-Arg chloromethyl ketone

[0092] Tissue factor (TF) is the cellular receptor for factor FVIIa(FVIIa) and the complex is principal initiator of blood coagulation. Wehave studied the effects of FVIIa binding to TF on cell migration andsignal transduction of human fibroblasts that express high amounts ofTF. Fibroblasts incubated with FVIIa migrated towards a concentrationgradient of PDGF-BB at about one hundred times lower concentration thando fibroblasts not ligated with FVIIa. Anti-TF antibodies inhibited theincrease in chemotaxis induced by FVIIa/TF. Moreover, a pronouncedsuppression of chemotaxis induced by PDGF-BB was observed with activesite-inhibited FVIIa (FFR-FVIIa). The possibility was excluded thathyperchemotaxis was induced by a putative generation of FXa and thrombinactivity.

[0093] FVIIa induced the production of inositol-1,4,5-trisphosphate tothe same extent as PDGF-BB; the effects of FVIIa and PDGF-BB wereadditive. FFR-FVIIa did not induce any release ofinositol-1,4,5,-trisphosphate. The cellular migration response toPDGF-BB and FVIIa was totally blocked by a PLC-inhibitor, suggestingthat activation of PLC is important for the response. Thus, binding ofFVIIa to TF can independent of coagulation, modulate cellular responses,such as chemotaxis, and the catalytic activity of FVIIa is necessary.

[0094] TF is believed to exert a function in tumour cell metastasis, butthe mechanism is yet not known. However, Ott et al. very recentlyidentified actin-binding protein 280 (ABP-280) as a ligand for the TFcytoplasmic domain, providing a molecular pathway by which TF maysupport tumor cell metastasis. The molecular signals and the biologicalfunctions transduced by FVIIa/TF are, however, still poorly understood.

[0095] Human fibroblasts have a constitutive expression of TF. Thesecells also express receptors for platelet-derived growth factor (PDGF).PDGF induces in its target cells mitogenicity, actin reorganization anddirected cell migration (chemotaxis). We have previously shown thatPDGF-BB is an efficient chemotactic factor for human fibroblasts andthat the chemotactic response is mediated by the β-receptor class.Therefore, these cells were chosen to study putative signal transductionand cell migration induced by binding of FVIIa to TF.

[0096] Below we show for the first time a clear connection betweensignalling induced by FVIIa binding to TF and the cellular response to agrowth factor. We present data that in human fibroblasts the FVIIa/TFcomplex leads to a hyperchemotactic response to PDGF-BB. Furthermore,active site-inhibited FVIIa (FFR-FVIIa) in a dose-dependent waysuppressed the directed migration towards PDGF-BB. By the use ofspecific inhibitors to PLC and phosphatidylinositol 3′-kinase(PI3′-kinase) we also demonstrate that the hyperchemotactic responsetowards PDGF-BB induced by FVIIa/TF signalling is dependent uponphospholipase C (PLC) activity but independent of PI3′-kinase. FVIIa andPDGF-BB induced the production of inositol-1,4,5-trisphosphate (IP₃),one of the second messengers released after activation of PLC, in anadditive manner.

[0097] TF is constitutively expressed on the plasma membrane of manyextravascular cells, such as stromal fibroblasts in vascular adventitiaand in fibrous capsules of liver, spleen and kidney. Thus, expression ofTF is found at sites physically separated from the circulating blood andproviding a haemostatic envelope. Upon injury this barrier is thought toprotect the organism against bleeding. TF can, however, be induced inmonocytes/macrophages, vascular smooth muscle cells, endothelial cellsand in a number of tumour cells by a variety of agents, includingcytokines and growth factors. Induction at the transcriptional leveloccurs rapidly after stimulation, identifying TF as a growth-relatedimmediate early gene.

[0098] In this study we have investigated the role of TF as a signallingreceptor. We show that human fibroblasts with a constitutive expressionof TF upon ligand binding of FVIIa migrate towards extremely lowconcentrations of PDGF-BB. TF/FVIIa alone did not induce enhancedspontaneous migration, i.e. random migration. Thus, a combination ofintracellular signal transduction by FVIIa/TF and the growth factorPDGF-BB was necessary to achieve the motility response. Not only bindingto TF, but also the catalytic activity of TF/FVIIa was mandatory, sinceactive-site inhibited FVIIa did not elicit enhanced migration response.Furthermore, inhibitory monoclonal antibodies prevented enhancement ofthe chemotactic response by FVIIa. We also excluded that indirectsignalling occured due to FXa or thrombin, since TAP and Hirudin had noeffect on FVIIa/TF induced chemotaxis. We instead found that increasingconcentrations of FFR-FVIIa actively inhibited PDGF-BB inducedchemotaxis. Fibroblasts incubated with FFR-FVIIa showed completelynormal random migration. The inhibitory effect of FFR-FVIIa onPDGF-BB-induced chemotaxis was not observed in the presence of thecombination of anti-TF antibodies thereby ruling out the possibility ofFFR-FVIIa being toxic. The results suggest rather, that in cellsexpressing PDGF β-receptors and TF, the FVIIa/TF complex is ofimportance for the chemotactic response to PDGF-BB.

[0099] Our finding that FVIIa increases IP₃ production, and thepreviously reported data on FVIIa/TF induced Ca²⁺ oscillationsespecially in MDCK cells, strongly support the notion that PLC isactivated by FVIIa/TF signalling in a number of cells. In addition, thehyperchemotactic response in human fibroblasts to PDGF-BB induced byFVIIa/TF was blocked in a dose-dependent way by a PLC-inhibitor. We havepreviously found a similar hyperchemotactic response to PDGF-BB in PDGFβ-receptor Y934F mutant cells, which showed increased phosphorylationand activation of PLC-γ1. In these cells, the enhanced phosphorylationof PLC-γ1 correlated with a threefold higher IP₃ production compared towild-type PDGF β-expressing cells. The combination of FVIIa/TF andPDGF-BB induced about twofold increase in IP₃ production in humanfibroblasts. FVIIa/TF-induced IP₃ production, however, did not correlatewith phosphorylation of PLC-γ1. Tyrosine phosphorylation of PLC-γ2induced by FVIIa/TF cannot be excluded, but seems unlikely since theexpression of PLC-γ2 is very low in human fibroblasts. Moreover, theintracellular part of TF is not endowed with intrinsic protein tyrosinekinase activity. These results suggest that FVIIa/TF induces activationof β and/or δ PLC isozymes. In the assay for IP₃ release the cellculture medium was supplemented with 0.1% FBS containing only about 0.1nM FXa. We found that a concentration of more than 20 nM FXa isnecessary to induce IP₃ production. The mechanism by which β or δ PLCisozymes are activated remains to be elucidated. It is believed thatactivation involves the cooperation between TF and a membrane-associatedprotein.

[0100] Lately, the connection of TF with the cytoskeleton wasidentified. A molecular interaction between the cytoplasmatic domain ofTF and the actin filament-binding protein ABP 280 was shown.Furthermore, TF was found to be in close contact with actin and actinfilament-binding proteins, such as α-actinin and ABP280 in lamellipodiaand ruffled membrane areas in spreading epithelial cells. ABP 280, amember of the filamin subfamily, is required for normal function oflamellipodia and thus highly important for cell motility. PI3′-kinaseand PLC isozymes are implicated in chemotactic responses, such asmobilisation of actin-binding proteins. In previous studies we observedthat the PI3′-kinase pathway in PDGF-P receptor induced chemotaxis seemsless important in cells with over-expression and enhanced activity ofPLC-γ1. This was also the case for cells with FVIIa bonded to TF. Thisindicates that the magnitude of activation of PI3′-kinase and PLCisozymes will determine which of these pathways will dominate. Takentogether, our data show that cell migration is an important morphogenicfunction induced by FVIIa/TF signalling.

[0101] Chemotaxis plays a pivotal role in wound healing, angiogenesisand metastasis. Chemotaxis is also an important component in thedevelopment of atherosclerotic plaques. In these processes a variety ofcells express TF as well as PDGF and PDGF receptors. Restenosis is amajor complication following interventional procedure of obstructedarteries. PDGF has been implicated in the vessel wall's response(neointima formation) to mechanical injury by mediating the migrationand proliferation of smooth muscle cells and fibroblasts. We have shownnow for the first time that FVIIa binding to TF-expressing cells have anincreased chemotactic response to PDGF, which is independent of thecoagulation.

[0102] At present, not much is known about signaling pathway(s) that areinduced by proteolytically active VIIa and how the signals generated byVIIa could contribute to cellular processes. One possibility is thatFVIIa could induce the expression of growth regulators that actdownstream to induce cellular processes. To investigate thispossibility, in the present study, we have examined changes in thetranscriptional program in human fibroblasts in response to exposure toVIIa using a cDNA microarray that contain more than 8,000 individualhuman genes. We chose fibroblasts since these cells normally encounterserum, which contain growth factors and activated clotting factors inthe context of vascular injury due to physical (e.g., surgery) andpathophysiological conditions. The temporal program of gene expressionobserved in response to serum suggests that fibroblasts are programmedto interpret the abrupt exposure to serum nor as a general mitogenicstimulus but as a specific physiological signal. Characterization oftranscriptional activation in response to serum and growth factors alsosuggest that fibroblasts are an active participant in a conversationamong the diverse cells which collectively control inflammation,angiogenesis and wound healing.

[0103] cDNA microarray analysis with mRNA isolated from fibroblastsexposed to VIIa for 90 min shows upregulation of Cyr61. Northern blotanalysis confirmed the VIIa-induced expression of Cyr61 in fibroblasts.Although not as robust as in fibroblasts, VIIa also increases theexpression of Cyr61 in vascular smooth muscle cells. Induction of Cyr61expression is dependent on the FVIIa's catalytic activity since FVIIaifail to induce the expression of Cyr61 . Although factor Xa and thrombincould also induce the expression of Cyr61(data not shown), thesecompounds are not involved in FVIIa-induced expression of Cyr61 . Wefound no evidence for the generation of traces factor Xa and thrombin inour experimental system. Further, specific inhibitor of factor Xa andthrombin had no significant effect on the FVIIa-induced expression ofCyr61.

[0104] Cyr61 is an immediate-early gene that is transcriptionallyactivated by serum growth factors in fibroblasts. It encodes a secreted40 kDa, cysteine-rich and heparin-binding protein that associates withextracellular matrix and cell surfaces. Cyr61 is a member of an emerginggene family of conserved and modular proteins characterized by thepresence of an N-terminal secretory signal, followed by four modularstructural domains and 38 cysteine residues that are largely conservedamong members of the family. The protein family now consists of sixdistinct members, including Cyr61, connective tissue growth factor(CTGF) and an avian proto-oncoprotein, Nov (thus named as CCN family)(The CCN family is further described in Lau et al., Exp. Cell Res 248:44-57, 1999). Cyr61 protein is shown to (i) promote the attachment andspreading of endothelial cells in a manner similar to that offibronectin, (ii) enhance the effects of bFGF and PDGF on the rate ofDNA synthesis of fibroblasts and vascular endothelial cells (iii)promotes cell migration in both fibroblasts and endothelial cells.Recent studies show that Cyr61 acts as a ligand to integrin α_(γ)β₃, anadhesion receptor known to be involved in signaling that regulates anumber of cellular processes including angiogenesis and tumormetastasis. Purified Cyr61 protein was shown to stimulate directedmigration of human microvascular endothelial cell in culture through aα_(γ)β₃-dependent pathway and induce neovascularization in rat corneas.Furthermore, expression of Cyr61 in tumor cells promotes tumor growthand vascularization.

[0105] Based on the present data that show FVIIa induces Cyr61expression in fibroblasts, it is believed that FVIIa-induced Cyr61 isresponsible, acting through integrin α_(γ)β₃, for FVIIa-mediated cellmigration and tumor metastasis. Thus, Cyr61 links FVIIa-TF proteolyticalsignal to the integrin-signaling pathway. The observations that VIIacatalytic activity is required for migration of smooth muscle cells andtumor cells, and tumor metastasis are consistent with the otherobservation that FVIIa catalytic activity is required for the inductionof Cyr61.

[0106] In addition to Cyr61, VIIa could also induce other regulatorsthat could mediate FVIIa-induced biological responses. FVIIa binding tocell surface TF in pancreatic cancer cells was shown to selectivelyover-express uPAR gene. Earlier we have shown, using differentialdisplay technique, up-regulation of transcription of poly(A)polymerasegene in fibroblasts exposed to FVIIa. Although it would have beeninteresting to find out whether the cDNA microarray also showdifferential expression of PAP, the filter did not contain the PAP cDNA.In addition to Cyr61, our cDNA microarray also show differentialexpression of four other genes (see results), but the differentialexpression ratio was very close to the borderline significance. Since inpreliminary experiments we could not confirm their differentialexpression by Northern blot analysis and also the absence of anysuggestive relevant data on the ability of these gene products tomediate FVIIa-induced biological responses, we did not analyze theirexpression further. However, since CTGF is a structurally relatedmolecule to Cyr61 and elicit same biological responses as Cyr61, we haveexamined the expression of CTGF even though the relative ratio of CTGFexpression in FVIIa-treated sample vs the control sample in the cDNAmicroarray is 1.8(2 is a conservative estimate to be a real magnitude inthe assay). The data revealed that FVIIa also induced the expression ofCTGF and the kinetics of VIIa-induced expression of CTGF was similar tothat of Cyr61.

[0107] Although CTGF behaves very similar to Cyr61, subtle differencesexist between them. For example, (a) CTGF has shown to be mitogenic initself whereas Cyr61 has no intrinsic mitogenic activity but augmentsgrowth factor-induced DNA synthesis (b) Cyr61 stimulates chemotaxiswhereas CTGF stimulates both chemotaxis and chemokinesis (c) althoughboth Cyr61 and CTGF are ECM-associated signalling molecules, CTGF isshown to secrete into culture medium. Thus, it is possible that FVIIaregulates cellular functions locally via Cyr61 whereas acts at adistance from its site through the secretion of CTGF.

[0108] The regimen for any patient to be treated with FVIIa or anotherTF agonist or FVIIai or another TF antagonist as mentioned herein shouldbe determined by those skilled in the art. The daily dose to beadministered in therapy can be determined by a physician and will dependon the particular compound employed, on the route of administration andon the weight and the condition of the patient. An effective amount issuitably a daily dosage from about 5 μg/kg/day to about 500 μg/kg/day,preferably from about 10 μg/kg/day to 300 μg/kg/day, more preferred fromabout 15 μg/kg/day to 200 μg/kg/day, most preferred from about 20μg/kg/day to 100 μg/kg/day.

[0109] The FVIIa or another TF agonist or FVIIai or another TFantagonist should be administered in one single dose, but it can also begiven in multiple doses preferably with intervals of 4-6-12 hoursdepending on the dose given and the condition of the patient.

[0110] The FVIIa or another TF agonist or FVIIai or another TFantagonist may be administered intravenously or it may be administeredby continuous or pulsatile infusion or it may be administered directlyto the relevant site such as, for example, injected directly into atumour. FVIIa or another TF agonist or FVIIai or another TF antagonistis preferably administered by intraveneous injections and in an amountof about 100-100,000 units per kg body weight, and preferably in anamount of about 250-25,000 units per kg body weight corresponding toabout 5-500 μg/kg, a dose that may have to be repeated 2-4 times per 24hours.

[0111] Conventional techniques for preparing pharmaceuticalcompositions, which can be used according to the present invention are,for example, described in Remington's Pharmaceutical Sciences, 1985.

[0112] The compositions used according to this invention are prepared bymethods known per se by the skilled artisan.

[0113] In short, pharmaceutical preparations suitable for use accordingto the present invention is made by mixing FVII, FVIIa or another TFagonist or FVIIai or another TF antagonist, preferably in purified form,with suitable adjuvants and a suitable carrier or diluent. Suitablephysiological acceptable carriers or diluents include sterile water andsaline. Suitable adjuvants, in this regard, include calcium, proteins(e.g. albumins), or other inert peptides (e.g. glycylglycine) or aminoacids (e.g. glycine, or histidine) to stabilise the purified factorVIIa. Other physiological acceptable adjuvants are non-reducing sugars,polyalcohols (e.g. sorbitol, mannitol or glycerol), polysaccharides suchas low molecular weight dextrins, detergents (e.g. polysorbate) andantioxidants (e.g. bisulfite and ascorbate). The adjuvants are generallypresent in a concentration of from 0.001 to 4% w/v. The pharmaceuticalpreparation may also contain protease inhibitors, e.g. apronitin, andpreserving agents.

[0114] The preparations may be sterilised by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilising agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured in the form of sterilesolid compositions, which can be dissolved in sterile water, or someother sterile medium suitable for injection prior to or immediatelybefore use.

[0115] In different aspects the present invention concerns:

[0116] A method of regulating the expression of at least one gene in acell, comprising the steps of:

[0117] a) contacting said cell with factor VII (a) or a tissue factorantagonist

[0118] b) determining the expression of said gene in said cell.

[0119] The above method, wherein said cell is a human vascular cellexpressing tissue factor, including fibroblasts and smooth muscle cells.

[0120] The method, wherein said gene is selected from the groupconsisting of Cyr61 , CTFG, dopamine D2 receptor, EST incyte PD 395116or P2U nucleotide receptor.

[0121] The method, wherein said tissue factor antagonist is modifiedfactor VII (a) known as factor VIIai.

[0122] A method wherein the expression of said gene is enhanced.

[0123] A method wherein the expression of said gene is inhibited orminimized.

[0124] A method of enhancing the expression of said gene comprisingcontacting the cell with factor VIIa.

[0125] A method of inhibiting the expression of said gene comprisingcontacting the cell with modified factor VII known as FVIIai.

[0126] The method wherein said gene is EST PD674714.

[0127] A method for regulating cell migration, comprising the steps of:

[0128] a) contacting said cell with factor VIIa or a tissue factorantagonist;

[0129] b) determining the migration of said cell.

[0130] The method, wherein said cell is a human cell expressing tissuefactor, including fibroblasts, smooth muscle cells, tumour cells,haematopoietic cells and epithelial cells.

[0131] The method, wherein the tissue factor antagonist is modifiedfactor VIIa known as factor VIIai.

[0132] The method, wherein the modified factor VII is selected fromDansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethylketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Argchloromethylketone.

[0133] A method of enhancing cell migration, comprising contacting thecell with FVIIa or a tissue factor agonist.

[0134] A method of reducing or inhibiting cell migration, comprisingcontacting the cell with a tissue factor antagonist.

[0135] A method for inducing or enhancing wound healing in a patient,comprising administering to said patient an effective amount of apharmaceutical composition comprising Factor VIIa or a tissue factoragonist.

[0136] A method for inhibiting the invasiveness of tumour cellscomprising contacting said cells with an effective amount of a tissuefactor antagonist.

[0137] A method for inhibiting cell migration, invasion,migration-induced cell proliferation or angiogenesis in a patient havinga disease or condition associated with undesired cell migration,invasion, migration-induced cell proliferation or angiogenesis,comprising administering to said patient an effective amount of apharmaceutical composition comprising a tissue factor antagonist.

[0138] The method, wherein the disease or condition is primary tumourgrowth, tumour invasion or metastasis.

[0139] The method, wherein the tissue factor antagonist is modifiedfactor VII known as FVIIai.

[0140] Use of factor VIIa or a tissue factor antagonist for themanufacture of a medicament for regulating cell migration.

[0141] Use, wherein factor VIIa is used for the manufacture of amedicament for enhancing cell migration.

[0142] Use, wherein a tissue factor antagonist is used for themanufacture of a medicament for reducing or inhibiting cell migration.

[0143] The method, wherein the tissue factor antagonist is modifiedfactor VIIa known as factor VIIai.

[0144] Use, wherein the modified factor VII is selected fromDansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethylketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Argchloromethylketone.

[0145] The present invention is further illustrated by the followingexamples that, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES Example 1 Preparation of FVII

[0146] Human purified factor VIIa suitable for use in the presentinvention is preferably made by DNA recombinant technology, e.g. asdescribed by Hagen et al., Proc.Natl.Acad.Sci. USA 83: 2412-2416, 1986or as described in European Patent No. 200.421 (ZymoGenetics). FactorVIIa produced by recombinant technology may be authentic factor VIIa ora more or less modified factor VIIa provided that such factor VIIa hassubstantially the same biological activity for blood coagulation asauthentic factor VIIa. Such modified factor VIIa may be produced bymodifying the nucleic acid sequence encoding factor VII either byaltering the amino acid codons or by removal of some of the amino acidcodons in the nucleic acid encoding the natural FVII by known means,e.g. by site-specific mutagenesis.

[0147] Factor VII may also be produced by the methods described by Brozeand Majerus, J.Biol.Chem. 255 (4): 1242-1247, 1980 and Hedner andKisiel, J.Clin.Invest. 71: 1836-1841, 1983. These methods yield factorVII without detectable amounts of other blood coagulation factors. Aneven further purified factor VII preparation may be obtained byincluding an additional gel filtration as the final purification step.Factor VII is then converted into activated FVIIa by known means, e.g.by several different plasma proteins, such as factor XIIa, IX a or Xa.Alternatively, as described by Bjoern et al. (Research Disclosure, 269September 1986, pp. 564-565), factor VII may be activated by passing itthrough an ion-exchange chromatography column, such as Mono Q®(Pharmacia fine Chemicals) or the like.

Example 2 Preparation of FVIIai

[0148] Modified factor VII suitable for use in the present invention ismade, e.g. as described in International Publications Nos. 92/15686,94/27631, 96/12800 and 97/47651 ZymoGenetics/Novo Nordisk).

Example 3 Effects of FVIIa and FFR-FVIIa on the Chemotactic Response ofFibroblasts to PDGF-BB

[0149] Fibroblasts expressing active TF (FIG. 1A and FIG. 1B) wereincubated with 100 nM of FVIIa and seeded in the upper part of themodified Boyden chamber; while media containing 10% FBS and PDGF-BB atdifferent concentrations were added below the 150 μm micropore filter.The migration of the cells under conditions where medium containing 10%FBS without PDGF-BB was added below the filter was used as a measure ofrandom migration, and calculated as 100% migration. A significantmigration response was recorded at a concentration of 0.01 ng/ml PDGF-BBin cells stimulated by FVIIa compared to 1 ng/ml PDGF-BB for cells notligated with FVIIa, i.e. a 100-fold difference in concentration (FIG.2A). At 0.01-0.1 ng/ml PDGF-BB the migration response to FVIIa increaseddose dependently, starting at 25 nM and with a maximal effect at 50-100nM FVIIa (FIGS. 3A-D). No enhancement of random migration was observedafter activation with FVIIa. To test whether the proteolytically activeFVIIa was mandatory for the hyperchemotactic response to PDGF-BB,fibroblasts were also incubated with 100 nM FFR-FVIIa and assayed in theBoyden chamber in the same way (FIG. 2A). No increased chemotaxis wasobserved with FFR-FVIIa at low concentrations of PDGF-BB, 0.0-1 ng/ml.In contrast, a pronounced suppression of chemotaxis induced by 10-50ng/ml PDGF-BB was achieved by 100 nM FFR-FVIIa (FIG. 2A and 3A-D). Whenfibroblasts were preincubated with a mixture of three different TFantibodies and then with FVIIa or FFR-FVIIa, the migration response toPDGF-BB was identical to the response of fibroblasts without thepresence of ligand bonded to TF (FIG. 4A). An irrelevant monoclonal IgGantibody did neither prevent hyperchemotaxis induced by FVIIa nor theinhibition of the migration response induced by FFR-FVIIa (data notshown). The presence of the IgG antibodies or the three TF antibodiesdid not change random migration of the fibroblasts (data not shown).

Example 4 The Hyperchemotactic Response is not Mediated by FXa or byThrombin

[0150] Since FVIIa-induced signal transduction leading to thehyperchemotactic response to PDGF-BB was dependent on the catalyticactivity of FVIIa it was important to determine whether signallingoccurred directly or via FXa or thrombin generated by the FVIIa/TFcomplex. The enhanced migration response transduced by FVIIa/TF was notblocked by 0.2-10 μM Tick anticoagulant peptide (TAP), whichspecifically blocks the active site of FXa and prevents a furtheractivation of the coagulation cascade leading to thrombin formation(FIGS. 5A,5B). Neither addition of 5 U/ml Hirudin, a specific thrombininhibitor, had any effect on FVIIa/TF induced hyperchemotaxis (FIG. 6A).TAP and Hirudin did not influence the migration of fibroblast inresponse to PDGF without the presence of the ligand FVIIa (FIGS. 5A, 5B,6A). Thus, it is unlikely that the effect of FVIIa on chemotaxis ismediated via the activation of FX or thrombin.

Example 5 The Hyperchemotactic Response to PDGF-BB is Influenced byPLC-Dependent Pathways, but Independent of PI3′-Kinase.

[0151] Activation of PI3′-kinase has recently been shown to be importantfor PDGF β-receptor induced chemotaxis. Therefore, we investigatedwhether LY294002, a specific PI3′-kinase inhibitor, was able to blockthe chemotactic response induced by FVIIa/TF signalling. Fibroblastswere pretreated with LY294002 at indicated concentrations for 30 minutesat 37° C. before the addition of 100 nM FVIIa and assayed in the Boydenchamber as described. The concentration of PDGF-BB was kept constant at0.1 ng/ml throughout the assay, i.e. a very low concentration at whichFVIIa/TF induced a significant chemotactic response. LY294002 waspresent during the entire experiments. FIG. 7A shows that the migrationresponse to PDGF-BB mediated by FVIIa/TF-signalling was unaffected bythe inhibition of PI3′-kinase.

[0152] To investigate whether the FVIIa/TF-induced chemotactic responseinvolved the activation of phosphatidylinositol specific phospholipase C(PLC), we preincubated the fibroblasts with different concentrations ofU73122, a specific PLC-inhibitor, for 30 minutes at 37° C. before adding100 nM FVIIa; the cells were then patiented to the chemotaxis assay inthe presence of the inhibitor. A close analogue, U73343, without effectson PLC was used as negative control. The concentration of PDGF-BB waskept constant at 0.1 ng/ml also in these experiments. Pretreatment ofthe cells with the active PLC-inhibitor U73122 inhibited thehyperchemotactic response to 0.1 ng/ml PDGF-BB in a dose-dependent way,with a total inhibition at 1 μM (FIGS. 8A and 8B). No effect onchemotaxis was observed when the inactive analogue U73343 was used.

Example 6 FVIIa/TF Induce Activation of PLC

[0153] To further explore the importance of PLC activity for thehyperchemotactic response, we also analysed the direct effects ofFVIIa/TF on PLC activity in fibroblasts. Activation of PLC leads toproduction of two second messengers, inositol-1,4,5-trisphosphate (IP₃)and diacylglycerol. Fibroblasts were incubated with myo [³H] inositolovernight, and then with 100 nM FVIIa or FFR-FVIIa for 60 minutes,followed by incubation with or without PDGF-BB at indicatedconcentrations. Treatment with 100 nM FVIIa alone for 60 minutes inducedIP₃ release in fibroblasts at the same level as 10 ng/ml and 100 ng/mlPDGF-BB alone did (FIG. 9). Moreover, the combination of 100 nM FVIIaand 10 ng/ml or 100 ng/ml PDGF-BB doubled the IP₃ release. The activesite-inhibited FVIIa did not induce release of IP₃. These resultsclearly show that PLC is activated upon binding of FVIIa to TF.

Example 7 Phosphorylation of PLC-γ1 is not Enhanced by TF/FVIIaSignalling in Fibroblasts

[0154] In order to determine whether the PLC-γ1 isoform, which isactivated by certain tyrosine kinase receptors, was responsible for theincreased PLC activity induced by FVIIa/TF, tyrosine phosphorylation ofPLC-γ1 was studied. Fibroblasts were incubated in the absence orpresence of 100 nM FVIIa or FFR-FVIIa for one hour, followed by thestimulation with 0, 2, 10 or 100 ng/ml PDGF-BB. After 5 minutes ofincubation, the cells were lysed and PLC-γ1 was immunoprecipitated,separated by SDS-PAGE and immunoblotted with antiphosphotyrosineantibodies. Whereas a significant increase in tyrosine phosphorylationof PLC-γ1 was recorded with increasing concentrations of PDGF-BB,addition of FVIIa alone to the fibroblasts did not induce any tyrosinephosphorylation of PLC-γ1 (FIG. 10). Moreover, the combination of FVIIaand PDGF-BB at different concentrations did not induce any furtherphosphorylation compared to stimulation with PDGF-BB alone (FIG. 10).FFR-FVIIa had no effect on PLC-γ1 tyrosine phosphorylation (FIG. 10).Thus, other PLC isoforms than PLC-γ1 are responsible for the increasedPLC activity after FVIIa stimulation.

Example 8 Methods

[0155] Cell cultures. Human foreskin fibroblasts, AG1518 and AG1523 weregrown to confluence in Eagle's MEM supplemented with 10% fetal bovineserum (FBS). Before use, the cells were detached by trypsinization (2.5mg/ml for 10 min at 37° C.), washed in Hank's balanced salt solution,and resuspended in Eagle's MEM with 10% FBS or in Ham's mediumsupplemented with 0.1% FBS.

[0156] Proteins. Human FVIIa (Novo Nordisk A/S, Gentofte, Denmark), wasexpressed and purified as described²⁹. FFR-FVIIa (Novo Nordisk) wasobtained by blocking of FVIIa in the active site with D-Phe-L-Phe-L-Argchloromethyl ketone. Recombinant Tick anticoagulant peptide (TAP) waskindly provided by Dr. P. Vlasuk, Corvas (San Diego, Calif.). Hirudinwas purchased from Sigma. LY294002, U73122 and U73343 were obtained fromBiomol (Plymouth Meeting, Pa.). Anti-TF monoclonal antibodies, TF8-5G9,TF9-5B7 and MTFH-1 (Morrissey, J. H., Fair, D. S., Edgington, T. S.Monoclonal antibody analysis of purified and cell-associated tissuefactor. Thromb. Res. 52,247-261 (1988)) was a kind gift of Dr. James H.Morrissey, Oklahoma Medical Research Foundation. The phosphotyrosineantibody, PY99 was from Santa Cruz, Calif.

[0157] Flow cytometry. The surface expression of TF was analysed byimmunofluorescence with a flow cytometer (Coulter Epics XL-MCL, BeckmanCoulter, Fullerton, Calif., Coulter Electronics, USA). The instrumentwas calibrated daily with Immuno-Check™ or Flow Check™ calibration beads(Coulter). For indirect immunofluorescence experiments AG1518 or AG1523fibroblasts were washed twice with PBS containing 0.1% bovine serumalbumin (BSA), incubated for 30 minut˜ps on ice with afluorescein-isothiocyanate (FITC)-labelled anti-human TF monoclonalantibody (4508CJ, American Diagnostica, Greenwich, Conn. USA). Theanti-Aspergillus niger glucose oxidase monoclonal IgG1 (Dakopatts) wasused as a negative control. Mean channel fluorescence intensity (MFI)and percentage of positive cells were determined for each sample.

[0158] Determination of TF activity. The procoagulant activity of TF wasdetermined as described by Lindmark et al. (Lindmark, E., Tenno, T.,Chen, J., Siegbahn, A. IL-10 inhibits LPS-induced human monocyte tissuefactor expression in whole blood. Br. J. Haematol. 102, 597-604 (1998)).Briefly, aliquots containing 0.2×10⁵ AG1518 or AG1523 fibroblasts werewashed twice with PBS, placed in the wells of a 96-well microtitreplate(Nunc, Roskilde, Denmark). The procoagulant activity was measured in atwo-stage amidolytic assay where a chromogenic substrate, S-2222(Chromogenix, Mölndal, Sweden), is cleaved by FXa, which in turn isactivated from FX by the TF/FVIIa complex. A reaction mixture containingfinal concentrations of 0.6 mM S-2222, 2 mM CaCl₂ and coagulationfactors from the factor concentrate Prothromplex-T™ TIM4 (Baxter,Vienna, Austria) at a final concentration of 1 U/ml FVII and 1.2 U/mlFX, was added to the wells, and change in absorbance at 405 nm followinga 30 minutes incubation at 37° C. was determined. The measurements weredone in triplicate.

[0159] Chemotaxis assay. The migration response of fibroblasts wasassayed by means of the leading front technique in a modified Boydenchamber, as previously described (Siegbahn, A., Hanimacher, A.,Westermark, B., Heldin, C-H. Differential effects of the variousisoforms of platelet-derived growth factor on chemotaxis of fibroblasts,monocytes, and granulocytes. J. Clin. Invest. 85, 916-920 (1990) andNistér, M., Hammacher, A., Mellström, K., Siegbahn, A., Rönnstrand, L.,Westermark, B., Heldin, C-H. A glioma-derived PDGF A chain homodimer hasdifferent functional activities from a PDGF AB heterodimer purified fromhuman platelets. Cell 52, 791-799 (1988)). Micropore filters (pore size8 μm) were coated with a solution of type-1 collagen at room temperatureover night. The filters were air dried for 30 minutes immediately beforeuse. Human foreskin fibroblasts AG1523, were grown to confluence inEagle's MEM supplemented with 10% FBS. The cells were detached bytrypsinization (2.5 mg/ml for 10 minutes at 37° C.) and suspended inEagle's MEM with 10% FBS. The fibroblasts were incubated for 10 minuteswith or without FVIIa or FFR-FVIIa before assay. One hundred microlitersof the cell suspension (2×10⁵ cells/ml) was added above the filter ofthe Boyden chamber. PDGF-BB was diluted in assay media (Eagle's MEM with10% FBS) and added below the filter in the chamber. The cells wereincubated for 6 hours at 37° C. in a humidified chamber containing 95%air/5% CO₂. FVIIa or FFR-FVIIa were present during the entireexperiment. The filters were then removed, fixed in ethanol, stainedwith Mayer's Hemalun, and mounted. Migration was measured as thedistance of the two furthest migrating fibroblast nuclei of onehigh-power field (12.5×24) in focus. The migration distance in eachfilter was calculated as the mean of the readings of at least threedifferent parts of the filter. Experiments were performed with two tofour separate filters for each concentration of chemoattractant. Foreach set of experiments, the migration of fibroblasts toward the assaymedia served as control.

[0160] In cases when anti-TF monoclonal antibodies or inhibitors tocoagulation factors, TAP and Hirudin, were used, cells were preincubatedfor 10 minutes with these agents, then with or without FVIIa orFFR-FVIIa before the chemotaxis assay was performed. Antibodies, TAP orHirudin were also present during the entire chemotaxis experiment. Inexperiments where the effects on the migration response of differentinhibitors, LY294002, U73122 or U73343, were tested, cells werepreincubated for 30 minutes with the inhibitors at indicatedconcentrations, and the inhibitors were also present throughout theexperiments.

[0161] Assay for release of inositol trisphosphate (IP₃). Six-wellplates with semi-confluent cultures of AG1518 human fibroblasts, wereincubated over night ( approx. 20 hours) with 2 μCi of myo(³H) inositol(Amersham) in 2 ml Ham's F12 with 0.1% FBS. Medium was changed to Ham'sF12 with 0.1% FBS (containing 2 mM CaCl₂) and 20 mM LiCl and the cellswere incubated for 15 minutes at 37° C. Cells were then incubated in theabsence or presence of 100 nM FVIIa or 100 nM FFR-FVIIa for one hour.PDGF-BB (0, 10 or 100 ng/ml) was added and the incubation was continuedfor 10 minutes at 37° C. The IP₃ assay was performed as previouslydescribed by Eriksson et al. (Eriksson, A., Nånberg, E., Rönnstrand, L.,Engström, U., Hellman, U., Rupp, E., Carpenter, G., Heldin, C-H.,Claesson-Welsh, L. Demonstration of functionally different interactionsbetween phospholipase C-γ and the two types of platelet-derived growthfactor receptors. J. Biol. Chem. 270, 7773-7781 (1995)).

[0162] Assay for agonist-induced PLC-γ1 phosphorylation. Semi-confluentcultures of AG1518 were serum starved overnight (approx. 20 hours) inmedium containing 0.1% FBS, and then incubated in the absence orpresence of 100 nM FVIIa or FFR-FVIIa for one hour followed byincubation with 0, 2, 10 or 100 ng/ml PDGF-BB for 5 minutes at 37° C.Cells were lysed and PLC-γ1 was precipitated, essentially as previouslydescribed (Hansen, K., Johnell, M., Siegbahn, A., Rorsman, C., Engström,U., Wernstedt, C., Heldin, C-H., Rönnstrand, L. Mutation of a Srcphosphorylation site in the PDGF β-receptor leads to increasedPDGF-stimulated chemotaxis but decreased mitogenesis. EMBO J. 15,5299-5313 (1996)) with anti-PLC-γ1 antiserum generated by immunizingrabbits with a peptide corresponding to the carboxyterminus of bovinePLC-γ1 (Artega, C. L., Johnson, M. D., Todderud, G., Coffey, R. J.,Carpenter, G., Page, D. L. Elevated content of the tyrosine kinasesubstrate phospholipase C-γ1 in primary human breast carcinomas. Proc.Natl. Acad. Sci. USA 88, 10435-10439 (1991). Samples were separated bySDS-PAGE and immunoblotted with the phophotyrosine antibody PY99.

[0163] Statistical analysis. Data were analysed using the Statistica TMfor Windows package (StatSoft, Tulsa, Okla. USA). A Student's t-test fordependent samples was used to determine statistical significance betweendifferent data sets. P values of <0.05 were considered statisticallysignificant.

[0164] Proteins. Recombinant human VIIa, a gift from Novo Nordisk(Gentofte, Denmark), was reconstituted in sterile water at aconcentration of 1 to 1.3 mg/ml. The stock VIIa solutions were checkedfor contaminating trace levels of endotoxin using limulus amebocytelysate (Bio Whittaker) and none was detected (detection level 30 pg).Recombinant tick anticoagulant protein (TAP) was kindly provided byGeorge Vlasuk (Corvas, San Diego, Calif.) and recombinant hirudin wasobtained from either Sigma (St.Louis, Mo.) or Calbiochem (San Diego,Calif.). Purified human factor Xa and thrombin were, obtained fromEnzyme Research Laboratories (Southbend, Ind.).

[0165] cDNA microarray. WI-38 cells were cultured to 80% confluency andserum deprived for 24 hours to enter quiescent state as described above.The culture medium was replaced with fresh serum-free DMEM (supplementedwith 5 mM CaCl₂) and allowed to stabilize for 2 h in culture incubator.Then, the cells were treated with purified recombinant VIIa (5 μg/ml)for 90 min. At the end of 90 min treatment, total RNA was isolated fromuntreated (control) and VIIa-treated cells using Trizol (GIBCO BRL).Poly (A) RNA was purified by a double pass over Oligo Tex mRNA isolationcolumns as described in manufacturer's technical bulletin (Qiagen).Eight hundred ng (800 ng) of highly purified poly (A) RNA from thecontrol and VIIa-treated cells were sent for cDNA microarray analysisservice (Human UniGEM V microarray, Genome Systems Inc, St. Louis, Mo.).

[0166] Northern Blot Analysis. Total RNA was prepared using TRIZOLreagent from quiescent monolayer of WI-38 cells that were exposed toVIIa and other materials as described in Results. Northern blot analysiswas carried out using standard procedure. Briefly, 10 μg of total RNAwas size fractionated by gel electrophoresis in 1% agarose/6%formaldehyde gels and transferred onto the nitrocellulose membrane by acapillary blot method. Northern blots were prehybridized at 42° C. witha solution containing 50% formamide, 5×SSC, 50 mM Tris.HCl, pH 7.5, 0.1%sodium pyrophosphate, 1% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mMEDTA, 100 μg/ml denatured salmon sperm DNA and 1% BSA and hybridizedwith ³²P-labeled Cyr61 cDNA probe (106 cpm/ml). The hybridized membraneswere exposed to either Dupont NEF or Fuji RX X-ray film. Forquantification purposes, the membranes were exposed to phosphor screenfor 1 to 4 h, and the exposed screens were analyzed in a Phosphorlmger(Molecular Dynamics) using “Image-quant” software. To obtain meanvalues, the units (counts) obtained from different experiments werenormalized to an internal control (counts present in control-treatedsample).

[0167] Chromogenic Assay. WI-38 cells were cultured in 96-well cultureplate and made them quiescent as described above. After washing thecells, FVIIa (5 μg/ml) in 1,00 μg of calcium containing buffer was addedto the culture wells containing cells or wells coated with buffer (nocells). After 30 min incubation, 25 μg of chromogenic substrates forfactor Xa and thrombin, i.e., Chromozym X and Chromozym TH were added tothe wells. After 3 h of color development, the plate was read in amicroplate reader. As controls, cells were incubated with traceconcentrations of factor Xa (50 to 0.1 ng/ml) or thrombin (0.1 to 0.002U/ml). No differences were found in absorbance at 450 nm between VIIaadded to cells, or VIIa added to wells not containing cells. The readingwas lower than the readings obtained with lowest concentration of factorXa or thrombin and represents VIIa chromogenic activity.

Example 9

[0168] cDNA microarray. Quiescent fibroblasts were exposed to a controlserum-free medium or the serum-free medium supplemented with VIIa (5μg/ml) for 90 min (three T-75 flasks for each treatment). After thetreatment, total RNA was harvested and poly (A) RNA was isolated. Sixhundred ng of mRNA was labeled with either Cy3 or Cy5 fluorescence andthen hybridized to the UniGem Human V chip containing 8,000 sequenceverified ESTs, representing up to 5,000 known human genes (serviceperformed by Genome System Inc for a fee). The control plate, in whichknown concentrations of reference cDNA was spiked into the probegeneration reaction to measure sensitivity and monitor the reversetranscription reaction, purification determine hybridization efficiencyand overall view of the quality and performance of the assay indicatedthe success of hybridization process. Global analysis of experimentaldata revealed minimal differences in hybridization signals between thecontrol and VII-treated samples-Only a small number of genes showedmoderate differential expression. We found upregulation of 5 genes (3.5to 2-fold higher in VIIa treatment) whereas one gene was down-regulatedupon VIIa treatment (2.4-fold lower) (+/−2 is a conservative estimatefor determining the minimum magnitude of real ratios). The identity ofthe 3.5-fold upregulated gene was not revealed due to the proprietarynature. Other VIIa-upregulated genes are Cyr61 (2.5-fold), dopamine D2receptor (2.2-fold), EST Incyte PD 395116 (2-fold) and P2U nucleotidereceptor (2-fold). It is interesting to note that CTGF, a gene belongingto the Cyr61 family, was 1.8-fold higher in VIIa-treated cells comparedto control cells. The downregulated transcript in VIIa-treated cells wasEST PD674714. We selected Cyr61 for further analysis.

Example 10

[0169] Confirmation of differential expression of Cyr61. To validate thedata obtained in microarray, we have patiented the RNA samples from thecontrol and VIIa-treated cells (the same RNA samples that have been usedto prepare poly (A) RNA for probe generation in the microarray) toNorthern blot analysis and probed with radiolabeled Cyr61 cDNA. The datashow that Cyr61 cDNA probe hybridized to a single transcript(approximately 2.0 kb) of RNA isolated from the control and VIIa-treatedcells. However, the intensity of hybridization signal was much higher inRNA isolated from VIIa-treated cells (FIG. 1). Quantitation ofhybridization signal revealed that expression of Cyr61 was 2.8-foldhigher in cells exposed to VIIa over the control treated cells.

Example 11

[0170] Kinetics of VIIa-induced expression of Cyr61. To determine thekinetics of Cyr61 expression, quiescent fibroblasts were treated forvarying time periods with 5 μg/ml VIIa. Total RNA was extracted andpatiented to Northern blot analysis. As shown in FIG. 2, Cyr61expression was increased in time-dependent manner in VIIa-treated cells.The expression was peaked at about 45 min and thereafter declined to thebase level in 2 to 3 h. Since it had been reported that expression ofCyr61 in mouse fibroblasts after stimulation with serum and growthfactor was sustained for several hours (up to 8 to 10 h) beforerepression occurs, we have examined the effect of serum and PDGF onkinetics of Cyr61 expression in quiescent human fibroblasts, WI-38. Asshown in FIG. 2B, Cyr61 is expressed only transiently upon stimulationwith PDGF and become fully repressed 2 h after the addition of stimuli.Similar results obtained with serum-induced expression of Cyr61 (datanot shown).

Example 12

[0171] Factor VIIa-dose dependent induced expression of Cyr61. Todetermine dose-dependency of VIIa, quiescent fibroblasts were treatedwith varying doses FVIIa (0.1 to 5 μg/ml) for 45 min and then total RNAsamples from the cells were patiented to Northern blot analysis. Asshown in FIG. 3, treatment of fibroblasts with as low as 0.1 μg/ml FVIIawas sufficient to induce the expression of Cyr61 and a plasmaconcentration of FVII(a) (0.5 μg/ml, 10 nM resulted in a prominentresponse, close to the maximal.

Example 13

[0172] Factor VIIa-catalytic activity is required for Cyr61 induction.To test whether VIIa catalytic activity is required for the induction ofCyr61, WI-38 cells were treated with VIIa and active-site inactivatedFVIIa (FVIIai) for 45 min and the expression of Cyr61 was evaluated byNorthern blot analysis. As shown in FIG. 4, FVIIai failed to induce theexpression of Cyr61 suggesting the requirement of FVIIa proteolyticactivity. In this context, it may be important to point out that FVIIaiwas shown to bind cell surface TF with the same or higher affinity thanFVIIa. It is unlikely that VIIa-induced expression of Cyr61 in ourexperiments was the result of generation of down-stream coagulationfactors, FXa and thrombin. By using sensitive chromogenic assays, wefound no evidence for the generation of factor Xa and thrombin in ourexperimental system (detection sensitivity 10 pg). Further, the specificinhibitors of factor Xa and thrombin, i.e., tick anticoagulant proteinand hirudin, respectively, failed to abolish VIIa-induced expression ofCyr61 (FIG. 5).

Example 14

[0173] Involvement of transcriptional mechanism for the induction ofCyr61 mRNA steady-state levels by VIIa. To investigate whethertranscription is involved in VIIa-mediated increase in Cyr61 mRNAsteady-state levels, quiescent WI-38 cells were incubated withactinomycin-D (10 μg/ml) for 30 min before the addition of VIIa for 45min. As shown in FIG. 6, actinomycin-D inhibited the stimulator effectof VIIa. This finding indicates a transcriptional mechanism forinduction of Cyr61.

[0174] To investigate whether de novo protein synthesis is required forthe induction of Cyr61 nRNA by VIIa, WI-38 cells were pretreated withthe protein synthesis inhibitor cycloheximide before the cells wereexposed to VIIa for 45 min. As shown in FIG. 6, the stimulatory effectof VIIa was not blocked by cycloheximide. In fact, cycloheximidemarkedly increased the VIIa-induced Cyr61 mRNA steady-state levels.

1. A method for inducing or enhancing cell migration, comprising thestep of contacting said cell with a tissue factor agonist
 2. The methodof claim 1, wherein the tissue factor agonist is FVII or FVIIa.
 3. Amethod of reducing or inhibiting cell migration, comprising the step ofcontacting the cell with a tissue factor antagonist.
 4. The method ofclaim 3, wherein the tissue factor antagonist is modified FVII.
 5. Themethod of claim 1 or claim 3, wherein said cell is a human cellexpressing tissue factor, including fibroblasts, smooth muscle cells,tumour cells, haematopoietic cells, monocytes, macrophages andepithelial cells.
 6. The method of claim 5, wherein said cell furtherexpresses PDGF and PDGF receptors, especially PDGF beta-receptors. 7.The method according to claim 4, wherein the modified factor VII isselected from factor VII modified with Dansyl-Phe-Phe-Arg chloromethylketone, Phe-Phe-Arg chloromethylketone, Dansyl-D-Phe-Phe-Argchloromethyl ketone and D-Phe-Phe-Arg chloromethylketone.
 8. A methodfor inducing or enhancing wound healing in a patient, comprisingadministering to said patient an effective amount of a pharmaceuticalcomposition comprising Factor VIIa or factor VII or another tissuefactor agonist.
 9. A method for inhibiting or reducing cell migration,invasion, migration-induced cell proliferation or angiogenesis in apatient having a disease or condition associated with undesired cellmigration, invasion, migration-induced cell proliferation orangiogenesis, comprising administering to said patient an effectiveamount of a pharmaceutical composition comprising a tissue factorantagonist.
 10. A method according to claim 9, wherein the disease orcondition is primary tumour growth, tumour invasion or metastasis.
 11. Amethod according to claim 9, wherein the tissue factor antagonist ismodified factor VII.
 12. Use of a tissue factor agonist for themanufacture of a medicament for inducing or enhancing cell migration.13. Use according to claim 12, wherein the tissue factor agonist is FVIIor FVIIa or a combination thereof.
 14. Use of a tissue factor antagonistfor the manufacture of a medicament for reducing or inhibiting cellmigration.
 15. The use of claim 14, wherein the tissue factor antagonistis modified factor VII.
 16. Use according to claim 15, wherein themodified factor VII is selected from factor VII modified withDansyl-Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone,Dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Argchloromethylketone.
 17. A method of regulating the expression of atleast one gene in a cell, comprising the step of contacting said cellwith a tissue factor agonist or a tissue factor antagonist, underconditions that result in a measurable change in said expression. 18.The method of claim 17, wherein the tissue factor agonist is selectedfrom the group consisting of FVII, FVIIa, and combinations thereof. 19.The method of claim 17, wherein the tissue factor antagonist is modifiedFVII.
 20. The method of claim 19, wherein the modified factor VII isselected from the group consisting of factor VII modified withDansyl-Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethylketone,Dansyl-D-Phe-Phe-Arg chloromethyl ketone and D-Phe-Phe-Argchloromethylketone.
 21. The method of claim 17, wherein the gene is agene belonging to the CCN gene family.
 22. The method of claim 17,wherein said gene is selected from the group consisting of Cyr61, CTFG,dopamine D2 receptor, EST Incyte PD 395116 and P2U nucleotide receptor.23. The method of claim 21, wherein the gene is Cyr61 gene.